Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description of the Invention. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The family of guanine nucleotide-binding (G) protein-coupled receptors (GPCRs) has been estimated to be comprised of as many as 2,000 members, fully more than 1.5% of all the proteins encoded in the human genome, that are thought to regulate function of virtually every cell in the body. Furthermore, it has been estimated that more than 50% of the drugs in use clinically in humans at the present time are directed at GPCRs.
In general, GPCRs require agonist binding for activation. Constitutive (or agonist-independent) signalling activity in mutant receptors has been well documented but only a few GPCRs have been shown to exhibit agonist-independent activity in the wild type (or native) form. For example, native dopamine D1B and prostaglandin EP1b receptors have been found to possess constitutive activity (Tiberi and Caron 1994; Hasegawa et al. 1996). A number of GPCRs, for example, receptors for thyroid-stimulating hormone (Vassart et al. 1995), have been found to be mutated to exhibit agonist-independent activity and cause disease in humans. Experimentally, several single amino acid mutations have produced agonist independent activity. .beta.2 and .alpha.2 adrenergic receptors, for example, mutated at single sites in the third cytoplasmic loop show constitutive activity (Ren et al. 1993; Samama et al. 1994). In some cases, a large deletion mutation in the carboxy tail or in the intracellular loops of GPCRs has led to constitutive activity. For example, in the thyrotropin releasing hormone receptor a truncation deletion of the carboxyl terminus renders the receptor constitutively active (Nussenzveig et al. 1993; Matus-Leibovitch et al. 1995) and a smaller deletion in the second extracellular loop of the thrombin receptor causes constitutive activity (Nanevicz et al. 1995) also.
The finding that certain receptors exhibit constitutive activity has led to a modification of traditional receptor theory (Samama et al. 1993). It is now thought that receptors can exist in at least two conformations, an inactive conformation (R) and an activated conformation (R*), and that an equilibrium exists between these two states that markedly favors R over R* in the majority of receptors. In some native receptors and in the mutants described above, it has been proposed that in the absence of agonist there is a shift in equilibrium that allows a sufficient number of receptors to be in the active R* state to initiate signalling.
As many more GPCRs with constitutive activity are found, both native as well as mutated receptors, a newly recognized class of drugs termed negative antagonists (or inverse agonists) will become important therapeutic agents. Negative antagonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this activity. Negative antagonists appear to act by constraining receptors in an inactive state (Samama et al. 1994). Although first described in other receptor systems (Schutz and Freissmuth 1992), negative antagonism has been shown to occur with guanine nucleotide-binding (G) protein-coupled receptors (GPCRs), for example, opoid (Costa and Herz 1989; Costa et al. 1992), .beta..sub.2 -adrenergic (Samama et al. 1994; Pei et al. 1994; Chidiac et al. 1994), serotonin type 2C (Barker et al. 1994), bradykinin (Leeb-Lundberg et al. 1994), and D1B dopamine receptors (Tiberi and Caron 1994). Thus, a technology for identifying negative antagonists of native and mutated GPCRs has important predictable as well as not yet realized pharmaceutical applications. Furthermore, because constitutively active GPCRs are tumorigenic, the identification of negative antagonists for these GPCRs can lead to the development of anti-tumor and/or anti-cell proliferation drugs.